Podophyllotoxin

ABSTRACT

The invention relates to podophyllotoxins, uses thereof and methods of their production.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/EP02/13669, filed Dec. 3, 2002, and designating the U.S.

Lignans, such as podophyllotoxin and its metabolites/precursors are partof the phenylpropanoid pathway and are widely distributed throughout theplant kingdom. Some of them, in particular podophyllotoxin itself, areknown to have anticancer, antifungal and/or antimicrobial properties.Podophyllotoxin was first extracted from may apple (Podophyllumpeltaturm) and from Linum species, such as Linum album, Linum flavum andLinum nodiflorum as a resin and was used by physicians in the southernparts of the USA in the late 19 ^(th) century for the treatment ofgenital warts, the latter being associated with some parts of cancerthrough their etiology by human papillomavirus (HPV). Podophyllotoxinhas gained particular interest as the parent molecule ofchemotherapeutic drugs such as etoposide, teniposide and etopophos,which are inhibitors of topoisomerase II. At present, the demand forpodophyllotoxin outreaches the global supply by far, which has becomeinsufficient due to slow growth and overcollection of the wild plants.In order to compensate for the limited supply of podophyllotoxinattempts have been undertaken to cultivate cells of Podophyllum peltatumand Linum album which, to some extent have been successful. Thesemisynthetic derivatives, etoposide and teniposide are widely usedimportant anticancerdrugs, but they have several limitations, such aspoor water solubility, metabolic inactivation and the development ofdrug resistance. To overcome these limitation derivatives ofpodophyllotoxin have been synthesised in many laboratories (Yin et al.,acta pharm. Sinica 1993, 28,758-761, Wang et al., acta chem. Sinica1992, 50, 698-701, Chang et al., J. med. chem. 1994, 37446-442, Pelteret al., J. nat. prod. 1994, 57, 1598-1602). None of these, however, haveproved a substantial amelioration in terms of both efficacy as well asthe prevention of side effects.

Etoposide is a widely used highly effective anti cancer drug against abroad spectrum of tumors including paediatric cancers such as acutelymphatic lymphomas, rhabdomyosarcomas and neuroblastomas as well as inmost common adult cancers. It is also used in bone marrowtransplantation conditioning regimens. However, the therapeutic use ofetoposide is limited by its toxicity involving mainly myelosuppression.

One of the major challenges for successful chemotherapy in cancer ingeneral and high risk leukemia in particular is to overcome multidrugresistance. The majority of patients initially respond to treatment withcombinations of various chemotherapeutic agents. However,polychemotherapy may induce multidrug resistant (MDR) cell clones whichcontinue to proliferate in the presence of cytotoxic agents (Dalton W S.Mechanism of drug resistance in hematologic malignancies Semin Hematol.1997; 34:3-8). The reduction of chemosensitivity in such cell cloneswould require the administration of cytostatic agents in quantitiesexceeding the maximum tolerated dose in vivo. One of thebest-characterized resistance mechanisms in leukemias and carcinomas isthe drug extrusion mediated by p-glycoprotein, the product of themultidrug resistance-1 gene (MDR-1), which has been shown to beassociated with poor outcome (Hunault M, Zhou D, Delmer A, et al.Multidrug resistance gene expression in acute myeloid leukemia: majorprognosis significance for in vivo drug resistance to inductiontreatment. Ann Hematol. 1997; 74:65-71; Marie J P, Zhou D C, GurbuxaniS, Legrand O, Zittoun R. MDR1/P-glycoprotein in haematologicalneoplasms. Eur J Cancer. 1996; 32A:1034-1038.).

A variety of strategies have been developed to avoid or circumvent drugresistance since the introduction of poly-chemotherapy. Efforts toovercome established drug resistance in patients focus on (i)scheduling, i.e. prolonged low dose therapy such as antifolates inrelapsed leukemia or short term high dose administration, e.g.antifolates with subsequent folate-rescue in osteosarcoma, (ii)combination therapy with chemical sensitizers such as MDR-1 inhibitorsin the case of MDR-mediated resistance and (iii) combination ofchemotherapy with non-chemical sensitizers such as radiotherapy,hyperthermia or hyperbaric oxygen (Dalton W S. Mechanisms of drugresistance in hematologic malignancies. Semin Hematol. 1997; 34:3-8;Joel S P, Slevin M L. Schedule-dependent topoisomerase II-inhibitingdrugs. Cancer Chemother Pharmacol. 1994; 34 Suppl:S84-S88; Ishikawa T,Kuo M T, Furuta K, Suzuki M. The human multidrug resistance-associatedprotein (MRP) gene family: from biological function to drug moleculardesign. Clin Chem Lab Med. 2000; 38:893-897.)

Only few attempts have been made to directly modify the cytostatic agentin order to find analogues that actively evade drug resistancemechanisms such as a deaminated doxorubicin analogue (Solary E, Ling YH, Perez-Soler R, Priebe W, Pommier Y. Hydroxyrubicin, a deaminatedderivative of doxorubicin, inhibits mammalian DNA topoisomerase II andpartially circumvents multidrug resistance. Int J Cancer. 1994;58:85-94.), and beta-amino derivatives of etoposide (Zhang Y L, Guo X,Cheng Y C, Lee K H. Antitumor agents. 148. Synthesis and biologicalevaluation of novel 4 beta-amino derivatives of etoposide with betterpharmacological profiles. J Med Chem. 1994; 37:446-452; Zhang Y L, ShenY C, Wang Z Q, et al. Antitumor agents, 130, Novel 4 beta-arylaminoderivatives of 3′,4′-didemethoxy-3′,4′-dioxo-4-deoxypodophyllotoxin aspotent inhibitors of human DNA topoisomerase II. J Nat Prod. 1992;55:1100-1111.). Thus, possible solutions to MDR related failures includethe rational design of drugs which are not affected by MDR mechanismsand exhibit reduced systemic toxicity and increased anti-tumor potency.

Resistance against etoposide occurs at distinct cellular levels,involving downregulation of the target enzyme topoisomerase II,downregulation of either pro- or upregulation of anti-apoptoticmechanisms such as bcl-2, and increased metabolism and/or extrusion ofthe drug from the cell mediated by transport systems. The induction ofsuch transport systems frequently leads to cross resistance againstother cytostatic agents, as observed for MDR-1, MRP, or LRP mediatedmultidrug resistance (Gottesman M M. How cancer cells evadechemotherapy: sixteenth Richard and Hinda Rosenthal Foundation AwardLecture. Cancer Res. 1993; 53:747-754; Borst P, Evers R, Kool M,Wijnholds J. A family of drug transporters: the multidrugresistance-associated proteins. J Natl Cancer Inst. 2000; 92:1295-1302;Borst P, Evers R, Kool M, Wijnholds J. The multidrug resistance proteinfamily. Biochim Biophys Acta. 1999; 1461:347-357.) A major mechanism ofdrug resistance, documented to occur in hematologic malignancies, is theoverexpression of the MDR-1 gene product, P-glycoprotein. Therefore,attempts to overcome transport-system mediated drug resistance focusedthus far mainly on modulation of MDR-1 expression (Liu C, Qureshi I A,Ding X, et al. Modulation of multidrug resistance gene (mdr-1) withantisense oligodeoxynucleotides. Clin Sci (Colch ). 1996; 91:93-98.) orcoadministration of MDR-1 inhibitors such as cyclosporin (Sonneveld P,Durie B G, Lokhorst H M, et al. Modulation of multidrug-resistantmultiple myeloma by cyclosporin. The Leukaemia Group of the EORTC andthe HOVON. Lancet. 1992; 340:255-259.), verapamil (Joly P, Lallemand A,Oum'Hamed Z, Trentesaux C, Idoine O, Desplaces A. Effects of verapamiland S9788 on MDR-1 MRNA expression studied by in situ hybridization.Anticancer Res. 1996; 16:3609-3614.) or valspodar (Tai H L. Technologyevaluation: Valspodar, Novartis A G. Curr Opin Mol Ther. 2000;2:459-467.) all of which revealed limited efficacy in vitro and in vivo.These membrane transport proteins extrude a surprisingly wide range ofsubstrates with entirely different structures, possibly due to the factthat common metabolites such as glucuronide, glutathione or sulfaterather than the different drugs are specifically recognized (Zhu B T. Anovel hypothesis for the mechanism of action of P-glycoprotein as amultidrug transporter. Mol Carcinog. 1999; 25:1-13.). Therefore, theseresistance mechanisms resemble the ubiquitin system, where a plethora ofentirely different proteins are “tagged” with ubiquitins in order to befirst recognized and then degraded by proteasomes. Thus drugmodifications that interfere with molecular “tagging” result in newmolecules which may not be cleared from multidrug resistant tumor cells.

Some attempts have been undertaken to synthesise derivatives ofetoposide which should allow a more specific targeting of etoposide tothe target tissues. EP0423747 reports the synthesis ofglycosyl-etoposide-prodrugs which by the action of tumor-specific enzymeconjugates can be cleaved into the effective etoposide drug and aglycosylic residue, whereby, because of the tumor specific enzymeconjugates, the drug is activated only at its preferred site of action.(cf. also U.S. Pat. No. 4,975,278). Shabat et al. (PNAS, vol. 98, 13,7528-7533) describe the synthesis of an antibody-prodrug system based onetoposide wherein the 4′-phenolic OH-group was masked by an aldolcarbamate compound. Such a prodrug alone, however did not show anyanti-tumour activity, unless it was combined with a catalytic antibody38C2, which activates the prodrug to yield etoposide. The prodrugsdescribed in Shabat et al. are only activated byretro-Michael/retro-aldol reactions which do not occur in nature. Onlyartificial enzymes, like the 38C2. catalytic antibody can catalyse theconversion. The handling and the use of such a prodrug is made moredifficult by the additional requirement of the co-application of acatalytic antibody which catalyses the conversion into the active drug.Therefore, none of the above mentioned derivative prodrugs have provenparticularly useful in the treatment of the above mentioned cancers.

Prodrugs of various antitumor agents have been synthesised in order toimprove their bio-availability, pharmacokinetics and aqueous solubility.WO99/30561 describes a nucleotide-based prodrug wherein the release andactivation of the drug component arises from the hydrolysis of thejunctional ester bond joining the nucleotide component to the drugcomponent.

U.S. Pat. No. 4,975,278 describes a method for the delivery of cytotoxicdrugs to tumor cells by the administration of a tumor-specificantibody-enzyme conjugate that binds to the tumor cells, and theadditional administration of a prodrug that is converted at the tumorside, in the presence of the antibody-bound enzyme, to an activecytotoxic drug. This concept has been used in conjunction withetoposide-4′-phosphate or 7-(2′-aminoethyl phosphate)-mitomycin. Againthis has been of limited utility so far.

WO94/13324 describes the conversion of drugs into prodrugs by convertingtheir corresponding functional groups into 1-O-alkyl-, 1-O-acyl-,1-S-acyl- and 1-S-alkyl-sn-glycero-3-phosphate derivatives. None of thereported prodrugs of WO94/13324 have proven particularly effective inthe treatment of cancers.

WO98/13059 reports on prodrugs comprising an amino-terminal cappedpeptide that is a substrate for a peptidohydrolase located on thesurface of a metastatic cell. The anticancerdrug typically used for thatpurpose is doxorubicine, taxol, camptothecin, mitomycin C oresperamycin. The peptidohydrolase that hydrolyses the substrate of theprodrug is typically cathepsin B.

U.S. Pat. No. 5,977,065 describes prodrugs of actinomycine D,doxorubicin, mytomycin C or nitrogen mustard arising from a reactionwith 4-nitrobenzylchloroformate.

European Patent Application EP 0320988 to Bristol-Myers Companydiscloses 4′-esters, 4′-carbonates and 4′-carbamates of4′-demethylepipodophyllotoxin glucosides for which a certain antitumoractivity in animals is reported. The compounds disclosed in EP 0320988are not capable of overcoming multidrug resistance.

Nicolaou (Nature 1993, 364, 464) and Niethammer et al. (Bioconj. Chem.2001, 3, 414) report on a paclitaxel prodrug blocked at the C7 hydroxylgroup with a dihydroxy propyl sidechain which can be hydrolyticallycleaved by a pH-dependent, slow-release mechanism. The resulting prodrugshowed some advantages in relation n to the parent drug in that it wasmore water soluble and could be used at a 3-fold higher maximumtolerated dose (MTD). Paclitaxel is an anticancer agent which isparticularly used in breast, lung and ovarian cancers. It is known topromote the irreversible polymerisation of tubulin thereby disruptingthe cell devision by cell cycle arrest in the premitotic G2 phase. Asecond cytotoxic mechanism of paclitaxel is to assist the induction ofTNF alpha, an event unrelated to the polymerisation of microtubules. Thepaclitaxel prodrugs described in the aforementioned publications areunstable in aqueous solution and hydrolyse spontaneously. Therefore theutility of the prodrugs reported in Niethammer et al. is very limited.So far, no prodrug has been reported in relation to etoposide whichshowed substantially improved efficacy and highly reduced side effectsin comparison to the parent molecule. Accordingly it has been an objectof the invention to provide for a prodrug of podophyllotoxins whichsubstantially reduces adverse reactions when administered to patient. Ithas also been an object of the present invention to provide for prodrugsof podophyllotoxins that are stable in aqueous solutions, yet do notrequire the application of catalytic antibodies for their conversationinto the active drug. It has furthermore been an object to provide forprodrugs of podophyllotoxins that allow for a slow release of the drugat the intended side of action, i.e. a tumor. It has also been an objectof the present invention to provide for prodrugs of podophyllotoxinsthat are capable of overcoming multidrug resistance commonly encounteredwith the parent molecule of the drug. Furthermore it has been an objectto provide for a method of preparing such prodrugs as well as apharmaceutical composition comprising such prodrugs as well as providingpotential uses of such prodrugs and such pharmaceutical composition.

This object is solved by a podophyllotoxin represented by formula I

wherein A is H or is selected from the group comprising carbohydrates,polyols,

ethylene glycol, propylene glycol, glycerol, penta-, erythritol,polyethyleneglycol and compounds, as represented by formula III below,wherein p is an integer from 2 to 100, andwherein B is represented by formula II—(C═X)—(Y)—(CH₂)_(n)-Z  (II)

-   -   wherein X is selected from the group comprising O, S and NR″, Y        is selected from the group comprising O, S, and NR″, wherein        R″=alkyl, aryl or H,    -   n is an integer of from 0 to 6; and    -   Z is a polyhydroxyalkyl group selected from the group comprising        ethylene glycol, propylene glycol, glycerol, pentaerythritol,        polyethyleneglycol and compounds represented by formula III

-   -   or Z is a polyhydroxyalkyl group, as defined above, which        additionally has a dioxolane group attached,    -   or Z is a dioxolane group,    -   or Z is selected from the group comprising targetting moieties        for mammalian receptors, antibodies, steroids, transferrin,        proteins and peptides having tumor cell associated receptor        finding function,

In one embodiment, the dioxolane group is selected from the groupcomprising 2,2-dialkyl-1,3-dioxolane, wherein each alkyl at the2-position is independently selected from the group comprisingunsubstituted and substituted methyl, ethyl, propyl, butyl, pentyl andhexyl.

In one embodiment, Z is a targeting moiety for a mammalian surfacemembrane receptor or for a mammalian nuclear receptor, or Z is anantibody, steroid, transferrin, protein or peptide having tumor cellassociated receptor binding function, wherein, preferably, Z is selectedfrom the group comprising steroids, growth factor receptor inhibitingproteins, peptides and non-peptide mimetics.

In one embodiment, A is selected from the group comprising compounds asrepresented by formula IV

wherein R₁ and R₂ are each C₁-C₁₀ alkyl; or R₁ and R₂ and the carbon towhich they are attached represent C₅-C₆ cycloalkyl; or R₁ is H and R₂ isselected from the group comprising C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₃₋₅cycloalkyl, furyl, thienyl, C₆₋₁₀ aryl, and C₇₋₁₄ aralkyl.

Preferably, R₁ is H and R₂ is methyl or thienyl.

In one embodiment, X is O and Y is O.

In another embodiment, X is O and Y is S.

In yet another embodiment, X is O and Y is NH.

In a preferred embodiment, the podophyllotoxin is selected from thegroup comprising

In another embodiment, it is selected from the group comprising

In yet another embodiment, it is selected from the group comprising

wherein Z is defined as in claim 1, wherein, preferably, thepodophyllotoxin is selected from the group comprising

The objects of the present invention are also solved by a method ofpreparing a podophyllotoxin, characterized in that a compound asrepresented by formula V

A being as defined before,

is reacted with a haloformate W—(C═X)—(Y)—(CH₂)_(n)-Z, wherein X, Y, Zand n are as defined before, and W being Cl, F, Br or I,

or characterized in that a compound as represented by formula V isreacted with phosgene or trichloromethylchloroformate, to yield a4′-phenol chloroformate intermediate,

-   -   said 4′-phenol chloroformate intermediate then being reacted        with an alcohol or thiol of the formula ZYH, to yield the        corresponding carbonate or thiocarbonate, Y═O or S, and Z being        as defined before, or    -   said 4′-phenol chloroformate intermediate then being reacted        with an amine of the formula HNR″Z to yield the corresponding        carbamate, R″ and Z being as defined before.

Preferably the compound as represented by formula V is reacted with acompound selected from the group comprising p-nitrophenylsoketalcarbonate, soketalchloroformate, and PEG-chloroformate.

In one embodiment of the method according to the present invention, theproduct resulting from the method as described before, is hydrolyzed.

The objects of the present invention are also solved by a pharmaceuticalcomposition comprising a podophyllotoxin according to the presentinvention and a pharmaceutically acceptable carrier.

The objects of the present invention are also solved by a use of apodophyllotoxin according to the present invention or of apharmaceutical composition according to the present invention for themanufacture of a medicament for the treatment of cell proliferativedisorders, wherein, preferably, the cell proliferative disorder is in achild, an adolescent or an adult.

In order to create hydrolytically activated prodrugs of etoposide VP-16as inactive precursor molecules which are activated by hydrolysis,derivatives of the 4′ phenolic hydroxy group were synthesised. Inprevious studies it could be demonstrated that this 4′ phenolic hydroxygroup is very important for the cytotoxic activity of etoposide since astable linker fused to that OH group decreased cytotoxicity by >3 logs(Shabat D, Lode H N, Pertl U, et al. In vivo activity in a catalyticantibody-prodrug system: Antibody catalyzed etoposide prodrug activationfor selective chemotherapy. Proc Natl Acad Sci U.S.A. 2001;98:7528-7533.). The prodrugs reported by Shabat, however, wereineffective in conferring cytotoxicity and did not show any anti-tumouractivity unless they were combined with the catalytic antibody 38C2which activates those prodrugs to yield the active drug. The prodrugsreported in Shabat et al. are only activated byretro-Michael-retro-aldol reactions which do not occur in nature, suchthat catalytic antibody or specifically tailored enzymes have to beadded that are capable of catalysing the conversion. In contrast theretothe prodrugs of the present invention are activated by a simplehydrolysis reaction, yet they are stable at a broad range of pH-valuesunder normal physiological conditions.

In contrast to paclictaxel, podophyllotoxins, in particular etoposide,are topoisomerase II inhibitors, with a wide range of applications inhuman ma lignancies, including solid tumors and leucemias which aredistinct from the field of applications of paclitaxel. For exampleetoposide is employed for the treatment of acute lymphatic leukemia,acute myeloic leukemia, neuroblastoma, and rhabdomyosarcoma. In contrastto the paclictaxel prodrugs that have been reported in theaforementioned publications (in particular Niethammer et al.) theprodrugs of the present invention are much more stable in aqueoussolutions under physiological conditions, yet can be easily hydrolysedby appropriate pH changes. Without wishing to be bound by theory it ispresently believed that one reason for that unexpected stability is thehydrophobic nature of the “southern” part of the molecule (i.e. at thephenol ring), in combination with the hydrophobic pocket which is formedat the 4′ position by the two neighbouring methoxy groups at the 3′ and5′ position, respectively.

Therefore, the rational design of hydrolytically activated prodrugs ofVP-16 involves the modification of the chemically reactive 4′ phenolichydroxy group which specifically tailors a desired effect. Depending onthe nature of the chemical modification, the activation of the prodrugcan be defined to occur at a desired pH dependent rate. The prodrugs ofthe present invention are stable under a wide range of pH values, yetcan be activated by an appropriate change of pH, as well as underphysiological conditions in the presence of naturally occurring enzymes,such as for example carboxyl esterases. This will be shown in moredetail below.

Therefore it was surprisingly found that by rational design,hydrolytically activated prodrugs of etoposide, that retain fullanti-tumor activity against multidrug resistant tumor cells in vitro andin vivo, can be produced.

As used herein the term “proteins and peptides having tumor cellassociated receptor finding function ” is meant to designate any proteinor peptide that is capable of binding a receptor that is associated withtumor cells.

“Targeting moieties for mammalian receptors” are groups that are capableof binding to mammalian receptors. “Growth factor receptor inhibitingproteins, peptides and non-peptide mimetics” is meant to designate anyprotein, peptide or non-peptide small molecule compound that is capableof inhibiting a growth factor receptor by any mechanism, in particularby binding thereto. “Etoposide and teniposide” is meant to designatecompounds of the formula

and any protonated form thereof. The term “functionality” is meant todesignate any chemical moiety allowing the molecule to which such moietyis attached, to undergo a chemical reaction.

Reference is now made to the figures, wherein

FIG. 1 shows a scheme of synthesis of ProVP-16I and II,

FIG. 2 shows the conversion of ProVP 16I to ProVP-16II and VP-16,

FIG. 3 shows cytotoxicity profiles for ProVP- 16I and II compared toVP-16,

FIG. 4 shows the effect of Pro VP-16I and II on multidrug resistantMOVP-3 cells,

FIG. 5 shows the induction of apoptosis by ProVP-16I and II in resistantcells, and

FIG. 6 shows the toxicity and anti-tumour response following ProVP-16IItherapy in mice.

In more detail, the figures are as follows:

FIG. 1 shows a Scheme of synthesis and activation of ProVP-16 I and II.The synthesis of ProVP-16 I involves the reaction of solketalchloroformate and VP-16 as described in Materials and Methods. CompoundProVP-16 II is synthesized from ProVP-16 I by acid hydrolysis with theelimination of 2,2 dihydroxypropane. The activation of ProVP-16 II toVP-16 occurs with the elimination of glycerol and carbon dioxide.

FIG. 2 shows the Conversion of ProVP16 I to ProVP-16 II and VP-16.ProVP-16 I was incubated in THF/2% HCl and samples were periodicallyanalyzed by HPLC at the indicated time points (A). Hydrolytic activationof ProVP-16 II was determined in phosphate buffered saline (PBS) a t thepH levels indicated (B). ProVP-16 I (3 mM) was incubated in PBSsolutions and samples were periodically analyzed by HPLC. The percentconversion was calculated from areas under the curve determined by peakintegration.

FIG. 3 shows Cytotoxicity profiles for ProVP-16 I and II compared toVP-16. The cytotoxic effect of ProVP-16 I and II was evaluated against apanel of cell lines by triplicate determinations of IC₅₀ concentrationsfor both prodrugs against each cell line (A). The cytotoxicity mediatedby the prodrugs relative to VP-16 was calculated according to IC₅₀VP-16÷IC₅₀ ProVP-16 I or II. Bars represent mean values ±SD. Thedifferences between ProVP-16 I or II and VP-16 were statisticallysignificant (p<0.01) for all cell lines except NXS2. Stars indicate celllines with amplified MDR-1 expression.

The slow release kinetics of cytotoxicity by hydrolytically activatedProVP-16 I was determined using Molt-3 cells (B). 10⁴ cells per wellwere incubated with increasing concentrations (10⁻¹⁰ M to 10⁻⁶ M) ofProVP-16 I and VP-16 in 96 well plates. At the time points indicated,cell viability was determined in triplicate by the XTT assay asdescribed in Materials and Methods. Per cent cell viability wascalculated from optical density measurements at 450 nm according to OD450_(sample)÷OD 450_(untreated)×100%. Results were plotted as asemilogarithmic function of drug concentration.

FIG. 4 shows the Effect of ProVP-16 I and I on multidrug resistantMOVP-3 cells. The newly generated VP-16 resistant MOVP-3 cell line wasanalyzed for MDR- 1 gene expression (A), resistance against VP-16induced cytotoxicity (B) and cross resistance to MDR-1 drugs which areknown substrates for p-glycoprotein (C). MDR-1 gene expression wasdetermined by RT-PCR analysis on total RNA isolated from MOVP-3 andMolt-3 cells, respectively (6A). Expression of GAPDH was used as acontrol for the integrity of the cDNA. Resistance of MOVP-3 cellsagainst VP-16 was calculated from IC50 concentrations according to IC50_(MOVP-3)÷IC50_(Molt-3)(n=3) and results compared to effects observedwith ProVP-16 I and II (6B). Differential findings between Molt-3 andMOVP-3 cells obtained with VP-16 were statistically significant(p<0.001) in contrast to ProVP-16 I and II (p>0.05). (6C)Cross-resistance of MOVP-3 cells against MDR-1 drugs (doxorubicin,melphalan, vinblastine and paclitaxel) was calculated from IC50 values(n=3) as described in 6B). Results for non-MDR-1 drugs (MTX, 5-FU,genistein, calicheamicin θ, ProVP-16 I) are shown as controls.Differential findings for MDR-1 drugs between MOVP-3 and Molt-3 cellswere all statistically significant (p<0.01) in contrast to non-MDR-1drugs (p>0.05).

FIG. 5 shows the Induction of apoptosis by ProVP-16 I and II inresistant cells. The effect of ProVP-16 I and II (5×10⁻⁷ M) on the cellcycle was analyzed in Molt-3 (A) and MOVP-3 (B) cells and results werecompared to VP-16 (5×10⁷ M). Cells were harvested at indicated timepoints (n=3), fixed, stained with propidium iodide and analyzed by FACSas described in Materials and Methods. Per cent cells in pre G1(apoptosic cells) were calculated from DNA histograms.

FIG. 6 shows the Toxicity and anti-tumor response following ProVP-16 IItherapy in mice. A/J mice (n=6) were injected i.p. with ProVP-16 II (20and 60 mg/kg) and VP-16 (20 mg/kg) on days 1, 3, 5, 7, 9, and 11 andwith VP-16 (60 mg/kg) on days 1,3 and 5 (A). The body weight wasdetermined for each animal over time and calculated as per cent of totalweight observed on day 0. An event as indicated by Kaplan Maier plots isdefined by loss of body weight greater than 20%.

The anti-tumor effect of ProVP-16 II therapy was determined in amultidrug resistant xenograft model (B,C). SCID mice (n=7) were injecteds.c. with 5×10⁶ MOVP-3 cells and primary tumors of 250 mm³ average sizewere established 55 days after inoculation. Treatment consisted of i.p.injections with Pro VP-16 II (45 and 15 mg/kg), VP-16 (15 mg/kg) andsolvent on days 55, 57, 59, 69, 71, 87 and 90 after tumor cellinoculation. Tumor growth was monitored by microcaliper measurements andtumor size was calculated as described in Materials and Methods (B).Differential findings between experimental groups of animals treatedwith ProVP-16 II (45 and 15 mg/kg) and control groups (solvent andVP-16) were statistically significant (p<0.001 after day 63) (B). Thebody weight of treated animals was determined over time and calculatedas per cent of body weight on day 57 (C).

At the end of the treatment experiment, remaining s.c. tumors wereremoved and analyzed by RT-PCR for- gene expression of MDR-1 except micetreated with 45 mg/kg with no residual tumor. Representative signals ofone tumor of each group is depicted and compared to MDR-1 expressingVCR-100 cells used as a positive control. The presence of a 229 or 127bp signals indicate expression of MDR-1.

The podophyllotoxins according to the present invention havesurprisingly proven to be particularly effective in mediatingcytotoxicity. Various derivatives of etoposide were synthesized andtheir activation mechanism via hydrolysis was clearly established (seeexamples that follow). Importantly, at physiological buffer conditions,the prodrugs remained stable. A prodrug of etoposide with a stablecarbamate linker to block the 4′ hydroxy group of VP-16 had already beenshown to be completely stable under physiological buffer conditions(Shabat D, Lode H N, Pertl U, et al. In vivo activity in a catalyticantibody-prodrug system: Antibody catalyzed etoposide prodrug activationfor selective chemotherapy. Proc Natl Acad Sci U S A. 2001;98:7528-7533.). In contrast to the hydrolytically activated prodrugs ofthe present invention, exemplified by ProVP-16 I and II, the carbamateprodrug was designed to be activated only by catalysis of a retro-aldolretro-Michael reaction mediated by catalytic antibody 38C2, a reactionwhich does not occur in nature. It has to be stressed that thiscarbamate prodrug as opposed to the prodrugs of the present inventionwas ineffective in mediating cytotoxicity against all tumor cell linesinvestigated, thus demonstrating a crucial role for the 4′ hydroxy groupin VP-16 as a biologically relevant center capable of inducingcytotoxicity. The active center is also blocked in the prodrugsdescribed here; however the prodrugs as comprised by the presentinvention with their hydrolytic activation mechanism do mediatecytotoxicity very effectively. The non-toxic nature of unconvertedProVP-16 I and II is shown by the slow release mechanism demonstrated inMolt-3 cells (FIG. 3) and by the absence of cytotoxic effects for theprodrugs up to 12 h of incubation is in contrast to VP-16. A steadyincrease of cytotoxic activity of both VP-16 derivatives over timeclearly demonstrated that the novel compounds of the present inventionare initially stable and non-toxic, but then become activated inside thetarget cells. The slow release mechanism observed in vitro also accountsfor the dramatically decreased systemic toxicity in mice, asdemonstrated for ProVP-16 II being tolerated a greater than 3 foldincrease in the maximum tolerated dose over the parental compound (FIG.6).

Further characterization of the prodrugs according to the presentinvention revealed a higher potency in a number of cancer cell linescompared to VP-16. Thus, in cells with amplified MDR-1 gene expression(VCR 100, ADR 5000 and SW480) more than 3 log greater efficacy of theprodrugs according to the present invention was observed (FIG. 4). Thisis an increase rarely achieved by MDR-1 modulators (Dalton W S.Mechanisms of drug resistance in hematologic malignancies. SeminHematol. 1997; 34:3-8; Sonneveld P, Durie B G, Lokhorst H M, et al.Modulation of multidrug-resistant multiple myeloma by cyclosporin. TheLeukaemia Group of the EORTC and the HOVON. Lancet. 1992; 340:255-259;Joly P, Lallemand A, Oum'Hamed Z, Trentesaux C, Idoine O, Desplaces A.Effects of verapamil and S9788 on MDR-1 mRNA expression studied by insitu hybridization. Anticancer Res. 1996; 16:3609-3614; Tai H L.Technology evaluation: Valspodar, Novartis A G. Curr Opin Mol Ther.2000; 2:459-467; Kang Y, Perry R R. Effect of alpha-interferon onP-glycoprotein expression and function and on verapamil modulation ofdoxorubicin resistance. Cancer Res. 1994; 54:2952-2958; Hofmann J,Gekeler V, Ise W, et al. Mechanism of action of dexniguldipine-HCl(B8509-035), a new potent modulator of multidrug resistance. BiochemPharmacol. 1995; 49:603-609.).

Furthermore, functional assays demonstrate that the prodrugs accordingto the present invention inhibit MDR-1 mediated substrate efflux.Consequently, it appears that the new prodrugs inhibit MDR-1p-glycoprotein function accounting for the excellent activity againstMDR-1 expressing cancer cell lines. Importantly, this dramatic in vitroeffect also translated into a long lasting regression of establishedprimary tumors in a drug resistant T-cell leukemia xenograft model invivo (FIG. 6). In this model, MDR-1 gene expression is stably amplifiedwith a 100× resistance against VP-16 in vitro (FIG. 4) resulting in thecomplete absence of a therapeutic effect by VP-16 at the maximumtolerated dose (FIG. 6). Interestingly, in such a challenging modelexhibiting an artificially high drug resistance, treatment with theprodrugs according to the present invention can induce a dramaticanti-tumor response (FIG. 6). This is of particular importance sincesuch a high MDR-1 amplification is rarely observed in patients followingpoly-chemotherapy even in relapsed malignancies (Beck J, HandgretingerR, Dopfer R, Klingebiel T, Niethammer D, Gekeler V. Expression of mdrl,mrp, topoisomerase II alpha/beta, and cyclin A in primary or relapsedstates of acute lymphoblastic leukaemias. Br J Haematol. 1995;89:356-363; Beck J F, Bohnet B, Brugger D, et al. Expression analysis ofprotein kinase C isozymes and multidrug resistance associated genes inovarian cancer cells. Anticancer Res. 1998; 18:701-705.).

An important finding of this study is the highly effective anti-tumorresponse observed in multidrug resistant models with the prodrugs of thepresent invention in vitro and in vivo, suggesting that therapy withthem will lead to a significant improvement over existing chemotherapywith ordinary podophyllotoxins.

The examples that follow are merely intended to illustrate, not to limitthe scope of the present invention.

EXAMPLE 1

Materials:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT), VP-16, solketal, organic solvents and phenazinemethosulfate (PMS) were obtained from Sigma-Aldrich (Deisenhofen,Germany). Cell culture reagents, restriction enzymes and other molecularbiology reagents were from Life Technologies (Karlsruhe, Germany).

Cells: Tumor cell lines were grown in RPMI, 10% FCS (Nalm-6, Reh,Molt-3, Jurkat, HL-60, K562, HeLa, CEM, A2780, SW480) or DMEM, 10% FCS(NXS2, SK-N-SH, HT-29) in the presence of 100 IU/mlpenicillin/streptomycin (P/S) and propagated under standard tissueculture conditions (5% CO₂, 37° C.). All cell lines were obtained fromATCC, Rockville, Md. except NXS2 which was previously described (Lode HN, Xiang R, Varki N M, Dolman C S, Gillies S D, Reisfeld R A. Targetedinterleukin-2 therapy for spontaneous neuroblastoma metastases to bonemarrow. Journal of the National Cancer Institute. 1997; 89:1586-1594.)and VCR100, ADR5000 and A2780 which were kindly provided by James Beck,Greifswald, Germany (Beck J, Handgretinger R, Dopfer R, Klingebiel T,Niethammer D, Gekeler V. Expression of mdrl, mrp, topoisomerase IIalpha/beta, and cyclin A in primary or relapsed states of acutelymphoblastic leukaemias. Br J Haematol. 1995; 89:356-363; Beck J F,Bohnet B, Brugger D, et al. Expression analysis of protein kinase Cisozymes and multidrug resistance associated genes in ovarian cancercells. Anticancer Res. 1998; 18:701-705.). Molt-3 cells were used togenerate an etoposide resistant subline MOVP-3 by continuous exposure toincreasing amounts of VP-16. After a time period of 6 months, MOVP-3cells were stable and propagated in the presence of 1 μM VP-16 and usedfor further in vitro and in vivo experiments.

Mice: Female A/J mice and FOX CHASE™ C.B-17/lcrCrl-scid BR mice wereobtained at 8 weeks of age from Charles River Laboratories, Sulzfeld,Germany. They were housed in the pathogen-free mouse colony at ourinstitution in groups of 8 mice. Mice were fed ad libitum on standardmouse laboratory chow. Animal experiments were performed according tothe German guide for the care and use of laboratory animals, i.e.“Tierschutzgesetz”.

Analytical chemistry of proetoposides: The synthesis of proetoposidesand analysis by HPLC has been previously reported Wrasidlo W, SchroederU, Bernt K, et al. Synthesis, hydrolytic activation and cytotoxicity ofetoposide prodrugs. Bioorg Med Chem Lett. 2002; 12. 557-560, which ishereby incorporated by reference in its entirety.

RNA isolation, reverse transcription and PCR amplification: Isolation oftotal cellular RNA, cDNA synthesis and RT-PCR-conditions were previouslydescribed (Lode H N, Xiang R, Varki N M, Dolman C S, Gillies S D,Reisfeld R A. Targeted interleukin-2 therapy for spontaneousneuroblastoma metastases to bone marrow. Journal of the National CancerInstitute. 1997; 89:1586-1594.). The amplification of human MDR-1 wasdone with sense 5′ GGA GAG ATC CTC ACC AAG CG 3′ and antisense 5′ GTTGCC AAC CAT AGA TGA AGG 3′ for 35 cycles (15 s 96° C., 30 s 60° C., 90 s72° C.) leading to a 229 bp fragment designated MDR-1. High sensitivitydetection was achieved by nested amplification of 1.0 μl MDR-1 after 21cycles using sense 5′ GCT CAG ACA GGA TGT GAG TT 3′ and antisense 5′ CTGGGT AAT TAC AGC AAG CC 3′ for 30 cycles to create a 127 bp fragment. ThecDNA integrity was tested by amplification ofglycerol-aldehyde-phosphate-dehydrogenase (GAPDH) with sense 5′ CGG GAAGCT TGT GAT CAA TGG 3′ and antisense 5′ GGC AGT GAT GGC ATG GAC TG 3′for 25 cycles leading to a 358 bp fragment. The specificity of allfragments was verified by sequencing.

Functional JC-1 assay: For staining, cells were washed twice andresuspended in PBS containing JC-1 monomer, as previously described(Legrand O, Perrot J Y, Simonin G, Baudard M, Marie J P. JC-1: a verysensitive fluorescent probe to test Pgp activity in adult acute myeloidleukemia. Blood. 2001; 97:502-508.). Briefly, 0.1 μM JC-1 monomer wasincubated at 37° C. for 15 minutes with 5×10⁵ cells/ml and incubated inthe presence and absence of etoposide and Pro-VP-16 I and II. Samplesincubated with 2 μM cyclosporin A were used as positive controls (datanot shown). Cell fluorescence was recorded using a FAC Sort flowcytometer (Becton Dickinson) and JC-1 signals were detected on theFL1-channel (530 nm filter) for analysis of the dye monomer.

Stable transfection of MDR-1: The MDR-1 cDNA was kindly provided in apUC based plasmid by C. Baum, University of Hamburg. For mammalianexpression, the MDR-1 cDNA was cloned into the bicistronic eukaryoticexpression plasmid pIRESpuro using NotI and BamHI restriction sites.pMDR-IRESpuro was transfected into Molt-3 cells by electroporation (960μF, 250V). Stable transfectants were selected with 300 ng/ml puromycin.MDR-1 expression was determined by FAC S-analysis (FAC S-calibur, BectonDickinson, Bedford, Mass.) using 1 μg/ml MDR- I specific mAb (C. Baum,University of Hamburg).

Cytotoxicity assay: Cytotoxicity was determined by the XTTtetrazolium/formazan assay as previously described (Scudiero D A,Shoemaker R H, Paull K D, et al. Evaluation of a solubletetrazolium/formazan assay for cell growth and drug sensitivity inculture using human and other tumor cell lines. Cancer Res. 1988;48:4827-4833.). Briefly, cells were seeded in 96 flat bottom well platesat a density of 10⁴/well in 100 μl media and exposed to drugconcentrations ranging from 10⁻⁴ to 10⁻¹² M. At the indicated timepoints (6-72 hr), cell viability was assessed by adding 50 μlXTT-reagent (1 mg/ml in serum free RPMI) activated with 0.2% v/v PMS(1.53 mg/ml in PBS) incubated at 37° C. for 4 h. Plates were analyzed ina Thermomax (Molecular Devices) micro plate reader at 450 nm. OD valueswere plotted as a function of drug concentration and the curves wereintegrated using the softmax software to obtain the IC₅₀ concentrationvalues.

Cell cycle analysis: Distinct phases of the cell cycle were determinedin a standard assay using propidium iodide. Briefly, cells were seededin 24 well plates (10⁵/well) and incubated with VP-16 or ProVP-16 I andII. At indicated time points, cells were harvested and fixed in 4.5 mlethanol (75%, −20° C.) for at least 12 h (4° C.). Cells were washed inPBS (pH 7.4) and resuspended in 250 μl PBS containing RNAse (0.3 mg/ml)and propidium iodide (50 μg/ml) and incubated in the dark (30 min, RT).The DNA histograms defining distinct phases of the cell cycle weresubsequently determined in duplicates by FACS analysis and averageresults were expressed as per cent.

Toxicity studies in mice: Stock solutions of 10 mM etoposide andproetoposide were prepared in 50:50 v/v cremophor:ethanol, and dilutedto the final concentration in PBS (pH 7.4). A/J mice, 8 to 10 weeks ofage were injected i.p. with 20 mg/kg or 60 mg/kg of either etoposide orproetoposide, or with solvent control (25% DMSO, 12.5% ethanol, 12.5%cremophor, 50% PBS), on day 1, 3, 5, 7, 9, and 11. VP-16 (60 mg/kg) wasonly injected on days 1,3 and 5. Body weights and survival weremonitored over time.

Anti-tumor effect in a T-cell leukemia xenograft model: Primary tumorswere induced by injection of 5×10⁶ MOVP 3 cells in 100 μl PBS (pH 7.4)into the skin of the left lateral flank of each SCID mouse. Establishedprimary tumors were palpable 55 days after injection. Mice (n=8) weretreated by intraperitoneal injection of solvent (25% DMSO, 12.5%ethanol, 12.5% cremophor, 50% PBS), VP-16 (15 mg/kg) and Pro VP-16 II(15 and 45 mg/kg) in a total volume of 200 μl. Each mouse received atotal of 7 injections on days 55,57,59,69,71,87,90 after tumor cellinoculation. Primary tumor size was determined over time by microcaliper measurements and volumes were calculated according to½×with²×length. Body weight was determined on a standard digital scale.

Statistics: The statistical significance of differential findingsbetween experimental groups of animals was determined by two-tailedStudent's t test. Findings were regarded as significant if two-tailed pvalues were <0.01.

Chemistry of etoposide prodrug activation: In order to establish thatProVP16-I and II are indeed prodrugs, we first determined theiractivation characteristics in vitro by HPLC (FIG. 1,2). Conversion ofProVP-16 I into ProVP-16 II and subsequent release of VP-16 follows atwo step activation mechanism (FIG. 1). First, ProVP-16 I converts toProVP-16 II with the elimination of 2,2-dihydroxypropane within 2 h withsome degradation (about 10%) of the glycoside moiety under -acidconditions (THF, 2N HCL) (FIG. 2A). Second, ProVP-16 II hydrolyses intoVP-16 under basic pH conditions with the elimination of glycerine (FIG.1,2B). In all experiments, conversion-time curves exhibited first orderkinetics. Importantly, under physiological buffer conditions (PBS, pH7.4, 37° C.), ProVP-16 II is stable with a conversion rate of <5% in 5up to 18 h. ProVP-16 I is completely stable with no measurableconversion in PBS (pH 7.4, 37° C.) and in contrast to ProVP-16 II, it isinert also under basic buffer conditions (pH <10.0). This unusualhydrolytic stability of ProVP-16 I is attributed to the hydrophobicnature of the entire southern region of this molecule and, to a lesserextent, also to steric hindrance from the two ortho methoxy groupssurrounding the carbonate moiety.

The utility of the prodrugs according to the present invention is alsoevident from the fact that they are activated, i.e. hydrolysed in thepresence of naturally occurring enzymes, but show stability in aqueoussolutions over a wide range of pH-values.

Activation of ProPV-16 I and II occurs in the presence of serum withconversion half lifes of 750.8 min and 56.1 min, respectively. Porcineliver caboxyl esterase also mediated conversion of ProVP16 I and II intoVP-16 with conversion half lifes of 14.2 min and 514.1 min,respectively. These findings clearly indicate enzymatic prodrugactivation at pH 7.4 by carboxyl esterases (Table 1).

TABLE 1 Kinetic parameters for hydrolysis of etoposide prodrugs media PHk_(obs) t_(1/2) compound (37° C. ± 0.5) (±0.1) (10⁻³-min⁻¹)^(a) (min)Prodrug I PBS buffer  5.0-10.0 no conversion — 2 11.8 8.333 ± 0.50 83.16 human serum 7.3 0.923 ± 0.046 750.8 esterase^(b) 7.3 48.89 ± 3.62 14.17 Prodrug II PBS buffer 5.0-7.3 no conversion — 3 8.0 3.201 ± 0.428216.49 8.8 7.142 ± 0.351 97.03 10.5 100.01 ± 12.72  6.93 human serum 7.312.36 ± 0.965 56.07 esterase 7.3 1.348 ± 0.081 514.10 ^(a)mean ± SD^(b)porcine liver carboxyl ester hydrolase

EXAMPLE 2

Activity of ProVP-16 I and II against leukemia and cancer cell lines:The cytotoxicity of these two proetoposides was tested against a panelof tumor cell lines using the XTT vital stain antiproliferation assay(FIG. 3A). For the majority of human tumor cell lines tested bothproetoposides were substantially more active than the parent compoundwith IC₅₀ values 1000 fold lower with SW480 colon carcinoma, 100-1000fold lower with HT-29 colon carcinoma, ADR5000 ovarian carcinoma andHL-60 pre B-cell leukemia, 10-100 fold lower with HeLa cervicalcarcinoma, A2780 ovarian carcinoma, K562 chronic myeloid leukemia,Jurkat T-cell leukemia, and SK-N-SH neuroblastoma, and 2-10 fold lowerwith Molt-3 T-cell leukemia, Reh and Nalm-6 pre B-cell leukemia, VCR100T-lymphoblastic leukemia and CEM T-lymphoblastic leukemia cells. Onlythree cell lines, NXS2 murine neuroblastoma (FIG. 3A), CHO Chinesehamster ovary, and Hamms human colon carcinoma cells responded equallywell to etoposide and proetoposides. Two cell lines, thedoxorubicin-resistant ADR5000 and the vincristine-resistant VCR100, bothwith amplified MDR-1 expression, revealed a higher cytotoxic potentialfor both proetoposides than for the non-resistant parental cell linesA2780 and CEM.

The time course of cytotoxic action of Pro-VP16 I was followed by usingMolt-3 T-cell leukemia cells (FIG. 3B). The results indicate a delayedonset of cytotoxic activity by ProVP-16 I, starting after 24 hr ofincubation. At that time point, the cytotoxic effect of parental VP-16was already almost completely established. The cytotoxic effect ofProVP16 I was completed 48-72 h after incubation (IC₅₀ 1.0×10⁻⁸) andexceeded that of VP-16 (IC₅₀ 6.5×10⁻⁸ M). Similar results were observedwith ProVP-16 II (data not shown). These findings indicate a slowrelease mechanism of cytotoxic activity from ProVP-16 I and II which isabsolutely consistent with the prodrug concept.

EXAMPLE 3

Effect of ProVP-16 I and II on multidrug resistant cells: Based on thefinding that ProVP-16 I and II are also more effective than VP-16 innatural MDR-expressing cell lines (FIG. 3), the question was addressedwhether these prodrugs could overcome artificial MDR in vitro. For thispurpose, the MDR-1 negative cell line Molt-3 was used to generate aresistant subclone MOVP-3 (FIG. 4A). MDR-1 mRNA expression in MOVP-3cells was determined by RT-PCR (FIG. 4A) while increased MDR-1 proteinexpression on the cell surface was established by FACS-analysis usingUIC2 monoclonal antibody. Extended gene expression analyses also lookingat MRP, LRP, Topisomerases I, IIα and IIβ as well as Bax and Bcl-2 (datanot shown) reveal that the only difference accounting for drugresistance in MOVP-3 cells in contrast to Molt-3 is expression of MDR-1.This was further confirmed by functional characterization of the sublinerevealed a 100-fold resistance against etoposide with IC₅₀ values of2×10⁻⁶ M for MOVP-3 and 2×10⁻⁸ M for Molt-3 cells (FIG. 4B). However,MOVP-3 cells remained almost fully sensitive towards ProVP16 I and IIwith no significant difference in proetoposide mediated cytotoxicitybetween parental Molt-3 and drug resistant MOVP-3 cells, with IC₅₀values of 2×10⁻⁸ M for both prodrugs and cell lines. Similar resultswere obtained in Molt-3 cells following stable transfection with MDR-1cDNA using pMDR-IRESpuro (Molt-3/MDR-1) that resulted in resistanceagainst etoposide, doxorubicin, taxol and vinblastine (data not shown).Molt-3/MDR-l cells also remained fully sensitive against ProVP-16 I andII. Controls stably transfected with empty vector (pIRESpuro) or avector containing GFP revealed no resistance to any of the drugs tested.

Furthermore the type of resistance in the MOVP-3 subline used wascharacterised by assessing the extent of cross resistance. MOVP-3 cellsdisplayed cross resistance against all MDR-1 type drugs (etoposide,doxorubicin, paclitaxel, vinblastine) in contrast to non-MDR-1 drugs(MTX, 5-FU, Genistein, Calicheamicin θ) (FIG. 4C). In summary, thesefindings clearly demonstrate that ProVP-16 I and II can overcome MDR- 1mediated multidrug resistance in vitro.

In a functional MDR-1 assay using JC-1 dye, an increased JC-1 efflux inMOVP-3 cells could be demonstrated in contrast to Molt-3 controls asindicated by a decrease in the FL-1 signal in the resistant subline incontranst to Molt-3 parental cells (FIG. 4D). This decrease wasabrogated by coincubation with 3×10⁻⁴ M Pro-VP-16 I (FIG. 4D) andinhibited MDR-1 mediated efflux over a broad concentration range down to10×10⁻⁶ M (data not shown). Interestingly, VP-16 used at equimolarconcentrations was ineffective in modulating MDR-1 function as indicatedby a JC-1 signal not different from PBS controls. These findings clearlyindicate that the prodrug design directly decreases MDR-1 mediatedsubstrate efflux.

In order to evaluate the cytotoxic mechanism mediated by ProVP-16 I andII in multidrug resistant MOVP-3 cells, the effect of these drugs on thecell cycle was analyzed at concentrations ranging from 10 μM to 1 μM andcompared to Molt-3 parental cells. Typical results obtained with 0.5 μMprodrugs are shown in FIG. 5. Specifically, asynchronous MOVP-3 (FIG. 5B) and Molt-3 (5 A) cells were incubated with VP-16, ProVP-16 I and IIfor 72 h. Periodically cells were subjected to cell cycle analysis atindicated time points. Results clearly indicate that both, Pro VP-16land II at 0.5 μM (FIG. 5B) as well as for the entire concentrationrange (10 nM-1 μM) (data not shown) are very effective in inducing asteady increase in the pre-G1 peak in MOVP-3 cells after 24 h,characteristic of apoptosis. In contrast to the prodrugs, VP-16 wasineffective to induce apoptosis, consistent with elimination of VP-16,but not ProVP-16 I and II, by MDR-1. Induction of apoptosis was alsodemonstrated in Molt-3 parental cells over the entire concentrationrange (0.5 μM, FIG. 5A). VP-16 was demonstrated to induce apoptosis inMolt-3 cells to a similar extent as ProVP-16 I and II. Overall, theseresults suggest that ProVP-16 I and II are no substrates for MDR-1.

EXAMPLE 4

In vivo toxicity and efficacy of ProVP-16 II in a multidrug resistantT-cell leukemia xenograft model: Based on the in vitro findings thatrevealed no differences between ProVP-16 I and II, ProVP-16 II wasselected for in vivo experiments because of higher water solubility.First, systemic toxicity of ProVP-16 II was determined in A/J mice (n=6)injected i.p. with VP-16 and ProVP-16 II (FIG. 6A). All mice receiving20 mg/kg VP-16 survived with an average weight loss of 10%. In contrast,5/6 mice treated with 60 mg/kg VP 16 showed a weight loss >20%. Thesefindings sharply contrast with the results obtained with ProVP-16 II. Inthat case administration of 20 and 60 mg/kg ProVP-16 II was welltolerated with no death in either experimental group. Only micereceiving 60 mg/kg ProVP-16 II revealed a transient weight loss of <10 %in contrast to 20 mg/kg Pro VP-16 II, which maintained stable averagebody weights. Thus, the maximum tolerated dose defined by a decrease inbody weight <20% was established at 20 mg/kg for VP-16 and at 60 mg/kgfor ProVP-16 II, consistent with a decrease of systemic toxicity of bythe prodrug design by at least a factor 3.

Second, the anti tumor effect of ProVP-16 II was determined in amultidrug resistant xenograft model of T-cell leukemia and compared toVP-16. Established primary tumors were induced by s.c. injection of5×10⁶ multidrug resistant MOVP-3 cells and tumor growth was followedover a time period of 105 days. Treatment was initiated 55 days aftertumor cell inoculation by i.p. injection at an average tumor size of 250mm³. The dose levels for VP-16 (15 mg/kg) and ProVP-16 II (15 and 45mg/kg) were selected based on results shown in FIG. 6A to further reducesystemic toxicity. Treatment with 45 mg/kg ProVP-16 II induced aregression of established primary tumors in 7/7 animals 10 days afterinitiation of treatment, which was stable for over 2 months (FIG. 6B).This treatment was well tolerated with a transient weight loss of only6% (FIG. 6C). This finding was in contrast to that observed in micetreated with 15 mg/kg VP-16, which showed no anti-tumor response andrevealed continuous primary tumor growth identical to control micetreated only with solvent. However, significant toxicity was observed inmice treated with 15 mg/kg VP-16 who exhibited a transient averageweight loss of 20% (FIG. 6C). In fact, mice receiving 15 mg/kg ProVP-16II showed no measurable weight loss (FIG. 6C) and presented with adramatic reduction in primary tumor growth in contrast to mice treatedwith the equivalent amount of 15 mg/kg VP-16. In order to determinewhether MDR-1 expression remained stable over the course of theexperiment, RNA was isolated from tumor explants at day 105 and alltumors investigated revealed an MDR-1 signal by RT-PCR (FIG. 6D).

The features of the present invention disclosed in the specification,the claims and/or in the accompanying drawings may, both separately andin any combination thereof, be material for realising the invention invarious forms thereof.

1. A podophyllotoxin it which is selected from the group consisting of


2. A podophyllotoxin represented by the formula: